In the last year, our work has covered several major areas that build on our previous work: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 20 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength via activation of PLC-g and Ca++ mobilization (Shaeffer et al. Science 1999; Shaeffer et al 2000, 2001, Nature Immunol.; Broussard et al. Immunity 2006; Horai et al. Immunity 2007; Gomez-Rodriguez et al., Immunity 2009, JEM 2014; Kapnick et al, J. Immunol. 2017, PNAS 2017). Confirming its importance, mutations of Itk have been described in a profound immunodeficiency associated with EBV-induced lethal lymphoproliferation. We have also shown that Itk is required for full activation of CD8 T cells and the final stages of degranulation of cytotoxic granules required for killing cellular targets (Kapnick et al, J. Immunol 2017). In related work, we used a variety of T cell receptor transgenic and mutant mice to probe requirements for terminating cytotoxic activity against a target, which may allow CTLs to proceed on and kill other targets, a process known as serial killing (Ritter, Kapnick et al, PNAS 2017). To follow up on these studies, as well as previous observations that Itk is required for Th2- and Th9-mediated responses, such as those associated with hypersensitivity and asthma (Gomez-Rodriguez, Nat. Comm. 2016; Schaeffer et al 2001), we have now developed a series of conditional Itk-mutant mice, including ones that can be inhibited acutely by designer inhibitors (Gomez-Rodriguez, in progress), providing potential models for Itk-based therapeutics. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work initially focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014; Cannons et al, Frontiers Immunol. 2018 in press). To further understand these defects, we have generated a mouse model and are using these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. Remarkably, these mice recapitulate multiple features of the disease. We are using them to dissect T and B cell-intrinsic components to these phenotypes, including autoimmunity, and have revealed a major role for the commensal microbiome in the development of autoantibodies (Preite et al. Nature Immunol. 2018 In Press), as well as a major role for PI3K in the development of longterm T cell memory required to prevent re-infection with viruses (Cannons et al, in progress). III. Regulation of Tfh cells and humoral immune responses: Another major focus of our work was SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects (Cannons et al. J. Immunol 2017; Panchal et al, Frontiers Immunol., 2018). Using gene-targeted mice we generated (Czar et al 2001), we showed that SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination (Crotty et al. Nature 2004; Cannons et al. 2006). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells (Qi et al, Nature 2008; Cannons et al, Immunity 2010; Lu et al, Immunity 2011), the critical helper T cell population required for providing signals to B cells for germinal center formation and long-term humoral immunity, a key feature of protective responses to most immunizations. Stemming from this work, we have continued to explore genes important for Tfh cell differentiation and function. Based on RNAseq evaluation of Tfh-specific gene-expression signatures, we found that the transcription factor TCF1, a component of the Wnt signaling pathway, is selectively expressed and required for Tfh cells in response to viral infection (Wu et al, Cell Reports, 2015). Our work has helped provide insight into the regulation of this important T helper cell population, which permits an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). To further uncover genes important for Tfh differentiation, we are using CRISPR-mediated mutagenesis in primary mouse T cells from Cas9 mice (Huang et al Curr. Prot In Immunol., In Press) to screen for genes affecting Tfh cells formation in vivo, as well as T cell adhesion (required for T cell interactions with other cells) in vitro. IV. In recent work, we have found that similar signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection, and that TCF1 marks a population of stem-like CD8 cells with remarkable transcriptional and metabolic overlap with Tfh cells (Wu et al, Sci Immunol 2016). In the last year, we have gone on to use single-cell RNAseq methods to learn more about this population during chronic infection and have now identified another transcription factor that is critical for the expansion and maintenance of these cells (Wu/Yao et al, in preparation). IV. To increase our ability to probe the immune system, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al PLOS One 2016 ; Huang et al. Curr. Protocols in Immunol. In press). We are using these tools to identify and probe function of genes involved in Tfh cell differentiation and in lymphocyte adhesion (Huang et al, in progress and Johansen et al, In progress).